The invention relates to the field of immunology, in particular to the field of cellular immunology.
It is also concerned with the area of organ transplantation, grafting of tissues or cells, especially bone marrow and possible immunological reactions caused by transplantation and/or grafting and bloodtransfusion.
Since the invention concerns a sex-related proteinaceous material, encoded in nature by a sex-related gene, the invention also relates to the areas of sex linked congenital aberrations, of embryonic selection techniques, in vitro fertilization techniques, vaccination and in ovo vaccination.
Bone marrow transplantation (BMT), one of the areas the invention is concerned with and the area from which the present invention originates, finds its application in the treatment of for instance severe aplastic anaemia, leukaemia and immune deficiency diseases.
In the early days of this technique many transplants failed through rejection of the graft by the host. Transplants that did succeed, however often led to an immune response by lymphocytes present in the graft against various tissues of the host (Graft versus Host Disease (GvHD)). It is now known that the GvHD response is mainly due to the presence of major H antigens which present a transplantation barrier. Therefor it is now routine practice to graft only HLA-matched materials (either from siblings or unrelated individuals) resulting in a much improved rate of success in bone marrow transplantation. However, despite this improvement, as well as improvements in pretransplantation chemotherapy or radiotherapy and the availability of potent immunosuppressive drugs, about 20-70% of the treated patients still suffer from GvHD (the percentage is age and bone marrow donor dependent). To avoid GvHD it has been suggested to remove the cells (mature T cells) causing said reaction from the graft. This however often leads to graft failure or to recurrence of the original disease. The cells responsible for GvHD are also the cells which often react against the original aberrant cells in for instance leukaemia (Graft versus Leukaemia response).
Since BMT is nowadays only carried out with HLA matched grafts, the GVHD which still occurs must be caused by another group of antigens. It is very likely that the group of so called minor H antigens (mHag), which are non-MHC encoded histocompatibility antigens (unlike the major H antigens) are at least partially responsible for the remaining incidence of GvHD.
mHag""s have originally been discovered in congeneic strains of mice in tumor rejection and skin rejection studies. In mice, the use of inbred strains has shown that mHag are encoded by almost 50 different allelically polymorphic loci scattered throughout the genome (24). In humans, mHag have been shown to exist, although their overall number and complexity remains uncertain. One of the better known, though unidentified minor histocompatibility antigens is the H-Y antigen. In the first report of H-Y as a transplantation antigen Eichwald and Silmser observed that within two inbred strains of mice, most of the male-to female skin grafts were rejected, whereas transplants made in other sex combinations nearly always succeeded (1). The term H-Y antigen was introduced by Billingham and Silvers (2) because the male specific antigen can function as a classical transplantation antigen responsible for homograft rejection.
Alloimmunity to human H-Y was first demonstrated in a female patient with aplastic anaemia who was given bone marrow from her HLA-identical brother. After a period of transient chimaerism the graft was rejected. At this time after grafting her lymphocytes showed unambiguously strong MHC restricted cytotoxic T cell (CTL) responses specific for male HLA-A2 positive target cells (3,4). The clinical case not only evidenced that H-Y can function as a transplantation barrier in man as well, but also that the recognition of the human male specific minor Histocompatibility antigen (mHag) was MHC restricted (4). The clinical relevance of H-Y as alloantigen is demonstrated especially in bone marrow transplantation (BMT) where sex-mismatch is one of the risk factors associated with rejection (3,4,5) or Graft-versus-Host-Disease (6,7). Sensitization to the H-Y antigen extends to organ transplantation (8-11), bloodtransfusion (12) and pregnancy (13), wherein MHC restricted T cell responses to the mHag H-Y in association with different MHC molecules are observed. To understand the impact of mHag H-Y on the outcome of organxe2x80x94and bone marrow grafting we earlier studied its tissue distribution. CTL mediated lysis of tissue-derived cell and cultured cell lines of several human tissues demonstrated an ubiquitous expression (11,14,16).
In search for the biological function of the gene encoding the mHag H-Y, our earlier studies analyzing lymphocytes from sex chromosomal abnormalities with our HLA restricted H-Y specific CTL clones revealed that absence of the mHag H-Y correlated with the XO and XX karyotype (17). Subsequent studies combining DNA, and functional expression with our CTL clones analyzing lymphocytes from individuals with Y chromosomal deletions, assigned the H-Y gene encoding the mHag H-Y to a portion of interval 6 (18), to a region covering the proximal segment of the Yq euchromatin, on the long arm of the Y chromosome (19).
Besides the role of H-Y as transplantation antigen, the human Y gene controlling the expression of the mHag H-Y is possibly also functioning as a gene controlling spermatogenesis. Agulnik et al. (20) recently identified a new murine Y chromosome gene, designated Smcy, controlling spermatogenesis as well the expression of the murine male specific mHag H-Y. The Smyc gene appears to be conserved on the Y chromosome in mouse, man and even in marsupials (20). It is notable that recent studies from our laboratories show recognition of the human HA-2 and H-Y peptides on non human primates cells, transfected with human class I genes, by our human HA-2 and H-Y specific class I restricted CTL clones (21).
Until recently, little was known about the molecular nature of the mHag gene products. Recent evidence was obtained revealing that the non-sexlinked human mHag HA-2 represents a short peptide originating from a member of the non-filament-forming class I myosin family (22). However, no information exists on the amino-acid sequence nor on the protein of the male specific mHag H-Y.
Aiming at the identification of the human H-Y peptide, we used the HLA-B7 restricted CTL clone xe2x80x9c5W4xe2x80x9d (12). Clone 5W4 originates from a female aplastic anemia patient who had received multiple transfusions (12,23).
Besides the HLA-B7 H-Y specific CTL clone, we earlier characterized HLA-A2 as well as HLA-A1 H-Y specific CTL clones (23).
We used a CD8 positive HLA-A2.1 restricted H-Y specific CTL clone, designated xe2x80x9c1R35xe2x80x9d (23). Besides, we also previously characterized a CD4 positive HLA-A2.1 restricted H-Y specific cytotoxic as well as proliferative T cell clone, designated as xe2x80x9cR416xe2x80x9d (41).
We aimed at identification of the human H-Y peptide recognized by the HLA-A2.1 restricted H-Y specific T cell clones IR35 and R416. The same methodology as applied for the identification of the HLA-B7 restricted H-Y peptide was used.
The invention thus provides a (poly)peptide comprising a T-cell epitope obtainable from the minor Histocompatibility antigen H-Y comprising the sequence SPSVDKARAEL (SEQ ID NO:1) or FIDSYICQV (SEQ ID NO:2) or a derivative of either of these having similar immunological properties.
The two sequences specified are encoded by the SMCY gene. The first sequence is the one found using the HLA-B7 restricted H-Y specific T-cell clone, The second is the one found using the HLA-A2.1 restricted clones.
The way these sequences are obtained is described herein. An important part of this novel method of arriving at said sequences is the purification and the choice of the starting material. Said novel method is therefor also part of the scope of this invention. However, now that the sequence is known, it is of course no longer necessary to follow that method, because the peptides can easily be made synthetically, as is well known in the art. Since routine techniques are available for producing synthetic peptides, it is also within the skill of the art to arrive at analogs or derivatives of the explicitly described peptides, which analogs and/or derivatives may have the same or at least similar properties and or activity. On the other hand analogs which counteract the activity of the explicitly described peptides are also within the skill of the art, given the teaching of the present invention. Therefor derivatives and/or analogs, be it of the same or different length, be it agonist or antagonist, be it peptide-like or peptidomimetic, are part of the scope of this invention.
A preferred embodiment of the present invention are the peptides with the sequences SPSVDKARAEL (SEQ ID NO:1) and/or FIDSYICQV (SEQ ID NO:2). This does not imply that other peptides are not suitable. This will for a large part depend on the application and on other properties of the peptides, which were not all testable within the scope of the present invention.
The peptides and other molecules according to the invention find their utility in that they may be used to induce tolerance of the donor immune system in H-Y negative donors, so that residual peripheral blood lymphocytes in the eventually transplanted organ or the bone marrow, as it may be do not respond to host H-Y material in an H-Y positive recipient. In this way GvHD may be prevented. On the other hand tolerance may be induced in H-Y negative recipients in basically the same way, so that upon receipt of an organ or bone marrow from an H-Y positive donor no rejection on the basis of the H-Y material occurs.
For tolerance induction very small doses can be given repeatedly, for instance intravenously, but other routes of administration may very well be suitable too. Another possibility is the repeated oral administration of high doses of the peptides. The peptides may be given alone, or in combination with other peptides, or as part of larger molecules, or coupled to carrier materials in any suitable excipients.
Further applications of the peptide or derivatives thereof lie in the prophylactic administration of such to transplanted individuals to prevent GvHD. This can be done with either agonists, possibly in combination with an adjuvant, or with antagonists which may block the responsible cells. This can be done with or without the concomittant administration of cytokines.
Furthermore the peptides or antibodies thereto can be used in so called xe2x80x9cmagic bulletxe2x80x9d applications, whereby the peptide or the antibody is coupled to a toxic substance to eliminate certain subsets of cells.
Diagnostic applications are clearly within the skill of the art. They include, but are not limited to H-Y typing, detection of genetic aberrancies and the like.
Other therapeutical applications of the peptide include the induction of tolerance to H-Y proteins in H-Y related (auto)immune diseases, such as possibly in Rheumatoid arthritis. On the other hand they may be used in vaccines in H-Y related (auto)immune diseases.
For the sake of illustration a number of applications is cited below.
The H-Y peptide or its derivatives can be used to prevent harmful reaction of the recipient towards the donor or vice versa; in all forms of transplantation i.e. organs, tissues and bone marrow. Assuming that residual donor peripheral blood lymphocytes (PBL)""s in the transplanted organ could react with and/or against host PBL""s and even could cause GvHD, the H-Y peptide could be used to induce tolerance in living organ (kidney, liver, gut, skin) of H-Y negative donors for H-Y positive patients. In bone marrow transplantation, the H-Y peptide (given alone or in combination with other peptides) can be used to induce tolerance in the living bone marrow donor. The peptide(s) can be given orally, intravenous or otherwise.
In all forms of organ (including cornea), tissue (including heartvalves and skin) and bone marrow transplantation with living or cadaveric donors, the H-Y peptide could be used to induce tolerance in H-Y negative recipients of organ and tissue transplants from H-Y positive donors. In case of bone marrow transplantation, tolerance must be induced in female donors for male recipients. The tolerance induction can be achieved by clinical application of the H-Y peptide systematically, i.v., locally, orally, as eye-drops.
The H-Y peptides could act in a non-allelic restricted manner (thus promiscuous) implicating that its applicability to inducing tolerance is not restricted to the HLA type of the female donors and female recipients and donors.
The H-Y peptides or their derivatives can be applied to generate reagents and/or medicine. They can be used as Graft-versus-Host disease and rejection prophylaxis administration to the transplanted individual either with or without adjuvant of
a) a H-Y peptide
b) H-Y peptide analogues, including left or right turning peptides
c) H-Y peptide antagonists
Usage of the H-Y sequence information to generate, for immunomodulatory purposes:
a) anti-idiotypic T cells
b) anti-idiotypic B cells
c) human monoclonal antibodies
The H-Y peptides or their derivatives can be used as a marker for sex linked congenital or other diseases.
They can be used for the generation of a genetic probe enabling screening for the congenital sex-linked disorders.
The genetic probe can be used for genetic counseling, population genetics and pre-natal diagnostic.
The defect can be repaired by genetic engineering.
The peptides and other molecules according to the invention can also be used for the production of anti-conceptive drugs.
Furthermore the peptides and other molecules according to the invention can be used for the production of cytotoxic T lymphoctes (CTL) with specificity for the H-Y sequence.
The H-Y specific CTL can be used for selection of male embryos in X linked recessive disorders.
The invented molecules can be applied to generate reagents and/or medicine for
a) determination of foetal erythrocytes in maternal circulation.
b) intra uterine diagnostics
c) use prior to implantation for in vitro fertilization.
d) determination of chimerism.
Veterinary applications include:
a) embryonic selection.
b) in vitro fertilization.
c) vaccination and in ovo vaccination
d) anti-conception.
On the basis of the peptides described herein genetic probes can be produced which can be used to screen for the gene encoding the protein. On the other hand such probes may be useful in detection kits as well. On the basis of the peptides described herein anti-idiotypic B cells and/or T cells and antibodies can be produced. All these embodiments have been made possible by the present disclosure and therefor are part of the present invention.
The techniques to produce these embodiments are all within the skill of the art.
Dose ranges of peptides and antibodies and/or other molecules according to the invention to be used in the therapeutical applications as described herein before are usually designed on the basis of rising dose studies in the clinic. The doses for peptides may lie between about 0.1 and 1000 xcexcg per kg bodyweight, preferably between 1 and 10 xcexcg per kg bodyweight.